1. Field of the Invention
The present invention relates to an epi-illumination apparatus for fluorescent observation and a fluorescence microscope having the same. In particular, the present invention relates to an epi-illumination apparatus for fluorescent observation for illuminating a sample that is marked with a plurality of fluorescence materials, and a fluorescence microscope having the same.
2. Description of the Related Art
Conventionally, fluorescent observations of a sample marked with a plurality of fluorescence materials have been conducted by using a fluorescence microscope or the like. The plurality of fluorescence materials adheres to respective different regions of the sample. Thus, based on the fluorescence occurring from the respective fluorescence materials, a fluorescence image of the sample can be captured to observe the plurality of different regions of the sample marked with the plurality of fluorescence materials simultaneously.
By the way, in order for respective fluorescence materials in a sample to produce fluorescence, the respective fluorescence materials must be excited by illuminations in appropriate narrow wavelength bands. The narrow wavelength bands of the illuminations appropriate for the respective fluorescence materials are typically different from each other. When the sample marked with a plurality of fluorescence materials is under fluorescent observation, the sample is thus irradiated with a plurality of illuminations of different narrow wavelength bands. Such a plurality of illuminations is usually generated by an excitation filter which transmits light in a plurality of predetermined, different narrow wavelength bands.
In general, fluorescence materials differ from each other in fluorescence efficiency (the ratio of the light intensity of fluorescence to the light intensity of illumination). Thus, when the sample is irradiated with a plurality of illuminations (in different narrow wavelength bands) of the same light intensities, the respective fluorescence materials produce fluorescence of different light intensities.
Then, the fluorescence image of the sample captured under the circumstances can show brighter images at regions where fluorescence materials of higher fluorescence efficiencies adhere to and darker images at regions where fluorescence materials of lower fluorescence efficiencies are adhere to. Such a mixture of brighter images and darker images in the fluorescence image of the sample makes it difficult to obtain a picture suited for fluorescent observation.
Consequently, in order to equalize the light intensities of the fluorescence occurring from the respective fluorescence materials of the sample, there has been proposed the method of adjusting the light intensity of plurality of illuminations for the sample to be irradiated with. For example, Japanese Patent No. 3093009 describes that an interference filter is arranged in the optical path of the illuminations between the light source and the excitation filter, and a rotating mechanism is provided to adjust the angle of incidence of the entrance beam to this interference filter.
In this configuration example, the spectrum of the illumination transmitted through the interference filter shifts and the spectrum of the illumination incident to the excitation filter shifts, by varying the rotation angle of the interference filter. As a result, a plurality of illuminations (in different narrow wavelength bands) generated by the excitation filter can be adjusted in light intensity.
In another method proposed, a plurality of interference filters having different transmission wavelength bands are prepared in advance. Any one of the interference filters is selectively put in the optical path of the illumination (between the light source and the excitation filter) according to the narrow wavelength band of the excitation filter, so that the spectrum of the illumination is modified to adjust the plurality of illuminations in the light intensity on the sample.
In the foregoing method of rotating an interference filter, however, the spectrum of the illumination transmitted through the interference filter can only be shifted within a small range. Thus, there has been the problem that the wavelength band allowing adjustments to the light intensities of the plurality of illuminations is narrow. In addition, each time the excitation filter is replaced with one having a different narrow wavelength band, the interference filter must also be replaced with another that has the range of shift suitable to the narrow wavelength band of the excitation filter. This means complicated operations and higher costs.
Moreover, in the foregoing method of switching interference filters, it is impossible to adjust the light intensities of the plurality of illuminations continuously. A single excitation filter requires a plurality of interference filters, which cause an increase in cost.